Plate layout

Shows the layout of samples, controls (e.g., negative controls (NC), inter-plate controls (IPC)), and special wells if any (e.g., sample controls (SC), bridge samples) on the 96 well assay plate.

Plate 1: Plate Layout
1 2 3 4 5 6 7 8 9 10 11 12
A SMI A1 Donor01 SMI A2 Donor02 SMI A3 Donor03 SMI A4 Donor04 SMI A5 Donor05 SMI A6 Donor06 SMI A7 Donor07 SMI A8 Donor08 SMI A9 Donor09 SMI A10 Donor10 SMI A11 PP01 rep01 SMI A12 IPC rep01
B SMI B1 Donor11 SMI B2 Donor12 SMI B3 Donor13 SMI B4 Donor14 SMI B5 Donor15 SMI B6 Donor16 SMI B7 Donor17 SMI B8 Donor18 SMI B9 Donor19 SMI B10 Donor20 SMI B11 PP01 rep02 SMI B12 IPC rep02
C SMI C1 Donor21 SMI C2 Donor22 SMI C3 Donor23 SMI C4 Donor24 SMI C5 Donor25 SMI C6 Donor26 SMI C7 Donor27 SMI C8 Donor28 SMI C9 Donor29 SMI C10 Donor30 SMI C11 PP01 rep03 SMI C12 IPC rep03
D SMI D1 Donor31 SMI D2 Donor32 SMI D3 Donor33 SMI D4 Donor34 SMI D5 Donor35 SMI D6 Donor36 SMI D7 Donor37 SMI D8 Donor38 SMI D9 Donor39 SMI D10 Donor40 SMI D11 PP01 rep04 SMI D12 IPC rep04
E SMI E1 Donor41 SMI E2 Donor42 SMI E3 Donor43 SMI E4 Donor44 SMI E5 Donor45 SMI E6 Donor46 SMI E7 Donor47 SMI E8 Donor48 SMI E9 Donor49 SMI E10 Donor50 SMI E11 PP02 rep01 SMI E12 NTC rep01
F SMI F1 Donor51 SMI F2 Donor52 SMI F3 Donor53 SMI F4 Donor54 SMI F5 Donor55 SMI F6 Donor56 SMI F7 Donor57 SMI F8 Donor58 SMI F9 Donor59 SMI F10 Donor60 SMI F11 PP02 rep02 SMI F12 NTC rep02
G SMI G1 Donor61 SMI G2 Donor62 SMI G3 Donor63 SMI G4 Donor64 SMI G5 Donor65 SMI G6 Donor66 SMI G7 Donor67 SMI G8 Donor68 SMI G9 Donor69 SMI G10 Donor70 SMI G11 PP02 rep03 SMI G12 NTC rep03
H SMI H1 Donor71 SMI H2 Donor72 SMI H3 Donor73 SMI H4 Donor74 SMI H5 Donor75 SMI H6 Donor76 SMI H7 Donor77 SMI H8 Donor78 SMI H9 Donor79 SMI H10 Donor80 SMI H11 PP02 rep04 SMI H12 NTC rep04

Read summary

Summary of the number and classification of reads for the assay run

Total reads Parseable Parseable Match Parseable Non-match Unparseable Total samples Total targets Total samples * targets Zero values
Plate 1 557,800,321 443,499,234 (79.5%) 320,863,871 (57.5%) 122,635,363 (22.0%) 114,301,087 (20.5%) 96 212 20,352 651 (3.2%)

Internal controls (IC)

Each sample of the assay is spiked with the same concentration of an internal control (IC). This is used both as part of a well normalization procedure and as a method for assessing the uniformity of the assay run.

mCherry (IC) summary
Zeros n (%) Total reads (%) Mean SD CV%
Plate1 0 (0.0%) 575,548 (0.2%) 5,995.3 3,314.8 55.3%

Inter-plate controls (IPC)

IPC samples are pooled plasma controls used both to normalize samples between experiments and to assess performance (e.g., precision) of the assay.

Note: %CV in this table does not exclude values below LOD. See Intra-plate normalization or Inter-plate normalization sections for %CV (before and after normalization) that excludes values below LOD.

Plate 1: IPC summary
Total count (%) Mean count per target Zeros n (%) CV% mean CV% median
Total 4,216,763 (1.3%) 4,972.6 20 (2.4%) 12.9% 11.8%
SMI_A12_IPC_rep01 954,338 (0.3%) 4,501.6 5 (2.4%)
SMI_B12_IPC_rep02 983,653 (0.3%) 4,639.9 5 (2.4%)
SMI_C12_IPC_rep03 1,079,936 (0.3%) 5,094.0 5 (2.4%)
SMI_D12_IPC_rep04 1,198,836 (0.4%) 5,654.9 5 (2.4%)
Median 1,035,337 (0.3%) 4,883.7 5 (2.4%)

Negative controls (NC)

NC wells are reactions where no sample input is provided (i.e., buffer only). These are used to assess Limit of Detection (LOD) for each target assay and to assess run quality (e.g., background levels).

Plate 1: NC summary
Total count (%) Mean count per target Max target count Missing n (%)
NC total 252,207.00 (0.1%) 297.4 184 (21.7%)
SMI_E12_NTC_rep01 60,385.00 (0.0%) 284.8 23,356 47 (22.2%)
SMI_F12_NTC_rep02 64,471.00 (0.0%) 304.1 24,663 47 (22.2%)
SMI_G12_NTC_rep03 69,440.00 (0.0%) 327.5 26,829 42 (19.8%)
SMI_H12_NTC_rep04 57,911.00 (0.0%) 273.2 22,004 48 (22.6%)
NC means 63,051.75 (0.0%) 297.4 24,213.0 24 (11.3%)

Sample controls (SC)

SC samples are pooled plasma controls from an independent pooled plasma source that is different from IPCs. They could be a pooled sample derived from all study samples, for example. They should be from the same source across all plates that will be analysed together. SCs are used for calculating intra- and inter-plate coefficient of variation (CV). They are necessary for obtaining an unbiased measure of inter-plate CV when IPC normalization is used.

Plate 1: SC summary
Total count (%) Mean count per target Zeros n (%) CV% mean CV% median
Total 9,478,631 (3.0%) 5,588.8 40 (2.4%) 17.4% 12.1%
SMI_A11_PP01_rep01 875,429 (0.3%) 4,129.4 5 (2.4%)
SMI_B11_PP01_rep02 798,857 (0.2%) 3,768.2 5 (2.4%)
SMI_C11_PP01_rep03 879,134 (0.3%) 4,146.9 5 (2.4%)
SMI_D11_PP01_rep04 924,573 (0.3%) 4,361.2 5 (2.4%)
SMI_E11_PP02_rep01 1,482,992 (0.5%) 6,995.2 5 (2.4%)
SMI_F11_PP02_rep02 1,663,813 (0.5%) 7,848.2 5 (2.4%)
SMI_G11_PP02_rep03 1,465,841 (0.5%) 6,914.3 5 (2.4%)
SMI_H11_PP02_rep04 1,387,992 (0.4%) 6,547.1 5 (2.4%)
Median 1,156,082 (0.4%) 5,453.2 5 (2.4%)

Heatmaps

Heatmaps show the percent relative to plate median for log2(total counts) and the specified internal control.

Quality control

Plate QC

Plate-specific QC criteria:

  • ICRead_CV - Coefficient of Variation of internal control signal across all samples (maximum = 50%)
  • IPCRead_CV - Coefficient of Variation of the total assay signal across all IPCs (maximum = 50%)
  • IPCTarget_CV - Median Coefficient of Variation of all IPC targets (maximum = 20%)
  • Detectability - Percentage of targets that are detectable (target is considered detectable if > 50% samples have signal > LOD) (minimum = 75%)
  • MinReads - Number of total Parseable Match reads (signal) for the experiment (minimum = 5e+05)

QC criteria are assessed either on unnormalized (raw) or IC-normalized (IC) data.

Plate 1: Plate QC Summary
QC Criterion Normalization QC Status QC Value QC Threshold
ICRead_CV raw Fail 55.3% 50%
IPCRead_CV raw Pass 10.5% 50%
IPCTarget_CV IC Pass 8.2% 20%
Detectability IC Pass 91.9% 75%
MinReads raw Pass 320,863,871 5e+05

Sample QC

Sample QC summary tables

Sample-specific QC criteria:

  • Detectability - Percentage of targets above LOD for a sample (minimum = 80%)
  • ICReads - Number of IC reads for a sample (minimum = 1000)
  • NumReads - Number of total Parseable Match (signal) reads for a sample (minimum = 5e+05)
  • IC_Median - % deviation from the overall plate median IC reads (within +/-30% of plate median)
Number of samples that fail each QC criterion (excludes IPC, SC, NC & bridge samples)
# of Samples Detectability ICReads NumReads IC_Median
Plate 1 80 1 0 0 1
Total 80 1 0 0 1
Samples that fail Detectability
Plate QC_fail_samples
Plate 1 SMI_G5_Donor65
Samples that fail IC_Median
Plate QC_fail_samples
Plate 1 SMI_G5_Donor65

Sample QC plots

Detectability

Target detectability is the percentage of samples that are above the limit of detection for that target. A target is considered “detectable” if it is above limit of detection in greater than 50% of samples. “Overall” detectability is the overall percentage of samples across all plates that are above LOD for a given target. Target detectability reported here excludes IPCs, SCs, NCs, and bridge samples.

Detectability summary

Plate 1
type # samples mean sd median min max # of detectable targets (%)
all 80 91.1 24.2 100 0 100 194 (91.9%)

Target detectability %

Plate 1
all
Activin AB 65.0
AGER 100.0
AGRP 100.0
ANGPT1 100.0
ANXA1 76.2
BDNF 100.0
BMP10 72.5
BMP15 0.0
BMP7 100.0
C1QA 100.0
CCL1 100.0
CCL13 100.0
CCL14 100.0
CCL15 100.0
CCL16 100.0
CCL17 100.0
CCL19 100.0
CCL2 100.0
CCL20 100.0
CCL22 100.0
CCL23 100.0
CCL24 100.0
CCL25 100.0
CCL28 100.0
CCL3 22.5
CCL4 100.0
CCL5 100.0
CCL7 98.8
CCL8 100.0
CD200 92.5
CD200R1 100.0
CD274 100.0
CD276 100.0
CD40 100.0
CD70 83.8
CD83 100.0
CD93 100.0
CEACAM5 100.0
CHI3L1 100.0
CLCF1 13.8
CLEC4A 100.0
CSF1 100.0
CSF2 0.0
CSF2RB 85.0
CSF3 100.0
CSF3R 100.0
CST7 100.0
CTF1 100.0
CTLA4 100.0
CTSS 100.0
CX3CL1 100.0
CXADR 100.0
CXCL1 100.0
CXCL10 100.0
CXCL12 100.0
CXCL13 100.0
CXCL14 98.8
CXCL2 100.0
CXCL3 100.0
CXCL5 100.0
CXCL8 100.0
CXCL9 100.0
EBI3 37.5
EGF 100.0
EPO 100.0
FAM3C 0.0
FAM3D 100.0
FASLG 100.0
FGF19 100.0
FGF2 93.8
FGF21 100.0
FGF23 100.0
FGF5 0.0
FLT1 100.0
FLT3LG 20.0
FTH1 100.0
GDF15 100.0
GDF2 100.0
GFAP 100.0
GRN 100.0
GZMB 100.0
HGF 100.0
IFNA1; IFNA13 98.8
IFNA2 52.5
IFNB1 95.0
IFNG 100.0
IFNL1 100.0
IFNW1 41.2
IL10 100.0
IL10RB 100.0
IL11 100.0
IL12B 100.0
IL12p70 95.0
IL12RB1 100.0
IL13 100.0
IL13RA2 100.0
IL15RA 100.0
IL16 100.0
IL17A 98.8
IL17B 100.0
IL17C 100.0
IL17F 98.8
IL17RB 100.0
IL18 100.0
IL18BP 100.0
IL18R1 100.0
IL19 98.8
IL1B 58.8
IL1R1 0.0
IL1R2 100.0
IL1RL1 100.0
IL1RN 98.8
IL20 100.0
IL22 61.3
IL23 98.8
IL2RA 100.0
IL2RB 100.0
IL32 91.2
IL33 98.8
IL34 100.0
IL35 2.5
IL36A 100.0
IL36G 91.2
IL39 6.2
IL3RA 100.0
IL4 100.0
IL4R 100.0
IL5 100.0
IL5RA 100.0
IL6 98.8
IL6R 100.0
IL7 100.0
IL7R 100.0
IL9 100.0
INHBA 87.5
INHBB 80.0
Inhibin A 25.0
Inhibin B 35.0
IRAK4 100.0
KDR 100.0
KITLG 98.8
LAG3 100.0
LAMP3 98.8
LCN2 100.0
LGALS9 95.0
LIF 98.8
LTBR 100.0
MDK 100.0
MERTK 100.0
MET 100.0
MICA 85.0
MICB 100.0
MIF 100.0
MMP10 100.0
MMP12 98.8
MMP3 100.0
MMP8 100.0
MMP9 100.0
MPO 100.0
MUC16 100.0
NCR1 100.0
NGF 100.0
NIC_1 0.0
NIC_3 0.0
OSM 100.0
PDCD1 100.0
PDGFA 100.0
PTX3 100.0
S100A12 100.0
S100A9 88.8
SCG2 98.8
SELE 100.0
SLURP1 100.0
SPP1 100.0
TAFA5 100.0
TGFB1 100.0
TGFB3 80.0
THPO 100.0
TIMP1 100.0
TIMP2 100.0
TLR3 100.0
TNF 86.2
TNFRSF11A 100.0
TNFRSF13C 100.0
TNFRSF14 100.0
TNFRSF17 100.0
TNFRSF18 100.0
TNFRSF1A 100.0
TNFRSF1B 100.0
TNFRSF4 100.0
TNFRSF8 100.0
TNFRSF9 100.0
TNFSF10 100.0
TNFSF11 92.5
TNFSF12 100.0
TNFSF13 100.0
TNFSF13B 98.8
TNFSF15 100.0
TNFSF4 100.0
TNFSF8 100.0
TNFSF9 100.0
TREM1 100.0
TSLP 100.0
VCAM1 100.0
VEGFA 37.5
VEGFC 100.0
VEGFD 88.8
VSNL1 100.0
VSTM1 97.5
WNT16 100.0
WNT7A 100.0

Target detectability boxplot

Plot shows target mCherry-IPC-normalized count distributions relative to LOD. For each target, log2(LOD) was subtracted from log2(count) for mCherry-normalized data.

Intra-plate normalization

%CV was calculated for each target (excluding internal controls) for the sample control replicates (if given) and IPCs. Only values above LOD were used to calculate %CV. If there are multiple plates, “Overall” represents the distribution for the target-specific mean intra-plate CV across all plates.

Intra-plate normalization

%CV was calculated for each target (excluding internal controls) for the sample control replicates (if given) and IPCs. Only values above LOD were used to calculate %CV.

Intra-plate %CV table – unnormalized

SC intra-plate %CV – unnormalized
mean sd median min max missing
Plate 1 15.6 13.8 11.2 2.05 106 10
IPC intra-plate %CV – unnormalized
mean sd median min max missing
Plate 1 12 3.67 11.7 1.48 30 14

Intra-plate %CV table – mCherry-normalized

SC intra-plate %CV – mCherry-normalized
mean sd median min max missing
Plate 1 15.1 13.7 11.2 1.23 105 10
IPC intra-plate %CV – mCherry-normalized
mean sd median min max missing
Plate 1 8.77 3.81 8.08 1.89 26.8 14

Intra-plate %CV boxplots by plate

Sample boxplots

Sample boxplots show distributions of the log2 counts for each sample, unnormalized and after mCherry + IPC normalization.

Sample correlation

Plot shows the Pearson correlation between samples. Heirarchical clustering is done using complete linkage.

Sample and target clustering

Hierarchical clustering of both samples and targets was done using a Euclidean distance metric and complete linkage. Data is log2 and mCherry-normalized; targets are centered and scaled.

Sample PCA